The major objective of this project is to define the cellular mechanisms by which hormones, neuro- transmitters, paracrines and growth factors interact to control hydrochloric acid secretion by parietal cells in the gastric mucosa. Gastroesophageal reflux and peptic ulcer disease are widespread clinical problems that result from or are exacerbated by gastric acid secretion. A better understanding of the mechanisms by which the major acid secretory agonists, histamine, acetylcholine and gastrin, and other factors control parietal cell acid secretion will allow for development of improved treatment and prevention of this disease. This research may also generate useful information on the stimulus- secretion coupling mechanisms and ion transport mechanisms that are applicable to other secretory cell types including, for example, those that are affected in diseases such as cystic fibrosis and cancer. There are two specific aims in this proposal. First, to continue ongoing cellular, biochemical and molecular studies to define the roles cAMP and Ca2??? signaling proteins in the regulation of HCI secretion. Second, to generate a lasp-1 knockout mouse model then study the effects of the deletion of this gene on acid secretory-related activities. Little is known about intracellular events that occur between initial agonist-receptor binding to the basolateral cell membrane and ultimate activation of the H, K-ATPase or proton pump at the apical cell membrane. Findings to date suggest, however, that in all cell types second messengers activate several different protein kinases and these kinases phosphorylate a variety of cellular proteins on serine, threonine and, rarely, on tyrosine residues. The characterization of the agonist-responsive proteins is essential if progress is to be made in our understanding of secretory control mechanisms in parietal cells and other cells as well. A wide range of methodologies will be used to address the specific aims of this proposal including parietal cell isolation and culture, cDNA transfection, site directed mutagenesis, in vitro measurement of acid secretory responses, digitized video image analysis, confocal microscopy and multiphoton microscopy to characterize acid secretory and intracellular signaling mechanisms in single parietal cells. [unreadable] [unreadable]